Dietary phytochemicals are known to exhibit a variety of anticarcinogenic properties. This study investigated the chemopreventive activity of blueberry extract in triple-negative breast cancer cell lines in vitro and in vivo. Blueberry decreased cell proliferation in HCC38, HCC1937, and MDA-MB-231 cells with no effect on the nontumorigenic MCF-10A cell line. Decreased metastatic potential of MDA-MB-231 cells by blueberry was shown through inhibition of cell motility using wound-healing assays and migration through a polyethylene terephthalate membrane. Blueberry treatment decreased the activity of matrix metalloproteinase-9 and the secretion of urokinase-type plasminogen activator while increasing tissue inhibitor of metalloproteinase-1 and plasminogen activator inhibitor-1 secretion in MDA-MB-231 conditioned medium as shown by Western blotting. Cell signaling pathways that control the expression/activation of these processes were investigated via Western blotting and reporter gene assay. Treatment with blueberry decreased phosphatidylinositol 3-kinase (PI3K)/AKT and NFκB activation in MDA-MB-231 cells, where protein kinase C and extracellular signal-regulated kinase (ERK) were not affected. In vivo, the efficacy of blueberry to inhibit triple-negative breast tumor growth was evaluated using the MDA-MB-231 xenograft model. Tumor weight and proliferation (Ki-67 expression) were decreased in blueberry-treated mice, where apoptosis (caspase-3 expression) was increased compared with controls. Immunohistochemical analysis of tumors from blueberry-fed mice showed decreased activation of AKT and p65 NFκB signaling proteins with no effect on the phosphorylation of ERK. These data illustrate the inhibitory effect of blueberry phytochemicals on the growth and metastatic potential of MDA-MB-231 cells through modulation of the PI3K/AKT/NFκB pathway. Cancer Res; 70(9); 3594-605.

Acquired resistance to either tamoxifen or aromatase inhibitors (AI) develops after prolonged treatment in a majority of hormone-responsive breast cancers. In an attempt to further elucidate mechanisms of acquired resistance to AIs, MCF-7aro cells resistant to letrozole (T+LET R), anastrozole (T+ANA R), and exemestane (T+EXE R), as well as long-term estrogen deprived (LTEDaro) and tamoxifen-resistant (T+TAM R) lines were generated. This is the first complete panel of endocrine therapy-resistant cell lines, which were generated as multiple independent biological replicates for unbiased genome-wide analysis using affymetrix microarrays. Although similarities are apparent, microarray results clearly show gene signatures unique to AI-resistance were inherently different from LTEDaro and T+TAM R gene expression profiles. Based on hierarchical clustering, unique estrogen-responsive gene signatures vary depending on cell line, with some genes upregulated in all lines versus other genes up-regulated only in the AI-resistant lines. Characterization of these resistant lines showed that LTEDaro, T+LET R, and T+ANA R cells contained a constitutively active estrogen receptor (ER)A that does not require estrogen for activation. This ligand-independent activation of ER was not observed in the parental cells, as well as T+EXE R and T+TAM R cells. Further characterization of these resistant lines was performed using cell cycle analysis, immunofluorescence experiments to visualize ER subcellular localization, as well as cross-resistance studies to determine second-line inhibitor response. Using this well-defined model system, our studies provide important information regarding differences in resistance mechanisms to AIs, TAM, and LTEDaro, which are critical in overcoming resistance when treating hormone-responsive breast cancers. [Cancer Res 2008;68(12):4910-8]

White button mushrooms (Agaricus bisporous) are a potential breast cancer chemopreventive agent, as they suppress aromatase activity and estrogen biosynthesis. Therefore, we evaluated the activity of mushroom extracts in the estrogen receptor-positive/aromatase-positive MCF-7aro cell line in vitro and in vivo. Mushroom extract decreased testosterone-induced cell proliferation in MCF-7aro cells but had no effect on MCF-10A, a nontumorigenic cell line. Most potent mushroom chemicals are soluble in ethyl acetate. The major active compounds found in the ethyl acetate fraction are unsaturated fatty acids such as linoleic acid, linolenic acid, and conjugated linoleic acid. The interaction of linoleic acid and conjugated linoleic acid with aromatase mutants expressed in Chinese hamster ovary cells showed that these fatty acids inhibit aromatase with similar potency and that mutations at the active site regions affect its interaction with these two fatty acids. Whereas these results suggest that these two compounds bind to the active site of aromatase, the inhibition kinetic analysis indicates that they are noncompetitive inhibitors with respect to androstenedione. Because only conjugated linoleic acid was found to inhibit the testosteronedependent proliferation of MCF-7aro cells, the physiologically relevant aromatase inhibitors in mushrooms are most likely conjugated linoleic acid and its derivatives. The in vivo action of mushroom chemicals was shown using nude mice injected with MCF-7aro cells. The studies showed that mushroom extract decreased both tumor cell proliferation and tumor weight with no effect on rate of apoptosis. Therefore, our studies illustrate the anticancer activity in vitro and in vivo of mushroom extract and its major fatty acid constituents.

Aromatase is the enzyme that converts androgen to estrogen. It is expressed at higher levels in breast cancer tissues than normal breast tissues. Grape seed extract (GSE) contains high levels of procyanidin dimers that have been shown in our laboratory to be potent inhibitors of aromatase. In this study, GSE was found to inhibit aromatase activity in a dosedependent manner and reduce androgen-dependent tumor growth in an aromatase-transfected MCF-7 (MCF-7aro) breast cancer xenograft model, agreeing with our previous findings. We have also examined the effect of GSE on aromatase expression. Reverse transcription-PCR experiments showed that treatment with 60 Mg/mL of GSE suppressed the levels of exon I.3-, exon PII-, and exon I.6-containing aromatase mRNAs in MCF-7 and SK-BR-3 cells. The levels of exon I.1-containing mRNA, however, did not change with GSE treatment. Transient transfection experiments with luciferasearomatase promoter I.3/II or I.4 reporter vectors showed the suppression of the promoter activity in a dose-dependent manner. The GSE treatment also led to the down-regulation of two transcription factors, cyclic AMP-responsive element binding protein-1 (CREB-1) and glucocorticoid receptor (GR). CREB-1 and GR are known to up-regulate aromatase gene expression through promoters I.3/II and I.4, respectively. We believe that these results are exciting in that they show GSE to be potentially useful in the prevention/treatment of hormone-dependent breast cancer through the inhibition of aromatase activity as well as its expression. (Cancer Res 2006; 66(11): 5960-7)

Resistance to endocrine therapy agents has presented a clinical obstacle in the treatment of hormone-dependent breast cancer. Our laboratory has initiated a study of microRNA regulation of signaling pathways that may result in breast cancer progression on aromatase inhibitors (AI). Microarray analysis of hormone refractory cell lines identified 115 differentially regulated microRNAs, of which 49 microRNAs were believed to be hormone-responsive. A group of microRNAs were inversely expressed in the AI-resistant lines versus LTEDaro and tamoxifen-resistant. We focused our work on hsa-miR-128a which was hormone-responsive and selectively up-regulated in the letrozole-resistant cell lines. Human miR-128a was predicted to target the TGFβ signaling pathway and indeed sensitivity to TGFβ was compromised in the letrozole-resistant cells, as compared to parental MCF-7aro. Human miR-128a was shown to negatively target TGFβRI protein expression by binding to the 3’UTR region of the gene. Inhibition of endogenous miR-128a resulted in re-sensitization of the letrozole-resistant lines to TGFβ growth inhibitory effects. This data suggests that the hormone-responsive miR-128a can modulate TGFβ signaling and survival of the letrozole-resistant cell lines. To our knowledge, this is the first study to address the role of microRNA regulation as well as TGFβ signaling in AI-resistant breast cancer cell lines. We believe that in addition to estrogen-modulation of gene expression, hormone-regulated microRNAs may provide an additional level of post-transcriptional regulation of signaling pathways critically involved in breast cancer progression and AI-resistance.

Serum- and glucocorticoid-inducible kinase 3 (SGK3) is a protein kinase of the AGC family of protein kinase A, protein kinase G, and protein kinase C and functions downstream of phosphatidylinositol 3-kinase (PI3K). Recent study revealed that SGK3 plays a pivotal role in Akt/protein kinase B independent signaling downstream of oncogenic PI3KCA mutations in breast cancer. Here we report that SGK3 is an estrogen receptor (ER) transcriptional target and promotes estrogen-mediated cell survival of ER-positive breast cancer cells. Through a meta-analysis on 22 microarray studies of breast cancer in the Oncomine database, we found that the expression of SGK3 is significantly higher (5.7-fold, P < 0.001) in ER-positive tumors than in ER-negative tumors. In ER-positive breast cancer cells, SGK3 expression was found to be induced by 17β-estradiol (E(2)) in a dose- and time-dependent manner, and the induction of SGK3 mRNA by E(2) is independent of newly synthesized proteins. We identified two ERα-binding regions at the sgk3 locus through chromatin immunoprecipitation with massively parallel DNA sequencing. Promoter analysis revealed that ERα stimulates the activity of sgk3 promoters by interaction with these two ERα-binding regions on E(2) treatment. Loss-of-function analysis indicated that SGK3 is required for E(2)-mediated cell survival of MCF-7 breast carcinoma cells. Moreover, overexpression of SGK3 could partially protect MCF-7 cells against apoptosis caused by antiestrogen ICI 182,780. Together, our study defines the molecular mechanism of regulation of SGK3 by estrogen/ER and provides a new link between the PI3K pathway and ER signaling as well as a new estrogen-mediated cell survival mechanism mediated by SGK3 in breast cancer cells.

Clinical trials have demonstrated the importance of aromatase inhibitor (AI) therapy in the effective treatment of hormone-dependent breast cancers. Yet, as with all prolonged drug therapy, resistance to aromatase inhibitors does develop. To date, the precise mechanism responsible for resistance to aromatase inhibitors is not completely understood. In this paper, several mechanisms of de novo/ intrinsic resistance and acquired resistance to AIs are discussed. These mechanisms are hypothesized based on important findings from a number of laboratories.To better understand this question, our lab has generated, in vitro, breast cancer cell lines that are resistant to aromatase inhibitors. Resistant cell lines were generated over a prolonged period of time using the MCF-7aro (aromatase overexpressed) breast cancer line. These cell lines are resistant to the aromatase inhibitors letrozole, anastrozole and exemestane and the anti-estrogen tamoxifen, for comparison. Two types of resistant cell lines have been generated, those that grow in the presence of Testosterone (T) which is needed for cell growth, and resistant lines that are cultured in the presence of inhibitor only (no T). In addition to functional characterization of aromatase and ERα in these resistant cell lines, microarray analysis has been employed in order to determine differential gene expression within the aromatase inhibitor resistant cell lines versus tamoxifen, in order to better understand the mechanism responsible for AI resistance on a genome-wide scale. We anticipate that our studies will generate important information on the mechanisms of AI resistance. Such information can be valuable for the development of treatment strategies against AI resistant breast cancers.

Exemestane-resistant breast cancer cell lines (i.e